Molecular chaperones maintain the appropriate folding and conformation of proteins and are crucial in regulating the balance between protein synthesis and degradation. They have been shown not only to play a vital role in the cellular stress response but also to be important in regulating many important cellular functions, such as cell proliferation and apoptosis (Jolly and Morimoto, 2000; Smith et al., 1998; Smith, 2001).
Heat Shock Proteins (HSPs)
Exposure of cells to a number of environmental stresses, including heat shock, alcohols, heavy metals and oxidative stress, results in the cellular accumulation of a number of chaperones, commonly known as heat shock proteins (HSPs). This effect is mediated by the transcription factor heat shock factor 1 (HSF1) and is termed the ‘heat shock response’ (Morimoto, 1998). Induction of HSPs protects the cell against the initial stress insult, enhances recovery and leads to maintenance of a stress tolerant state. It has also become clear, however, that certain HSPs may also play a major molecular chaperone role under normal, stress-free conditions by regulating the correct folding, degradation, localization and function of a growing list of important cellular proteins.
A number of multigene familes of HSPs exist, with individual gene products varying in cellular expression, function and localization. They are classified according to molecular weight, e.g., HSP70, HSP90, and HSP27. Exceptions to this nomenclature rule are a small subset of chaperones that were identified as glucose regulated proteins, e.g., GRP94 and GRP75.
Several diseases in humans can be acquired as a result of protein misfolding (reviewed in Tytell et al., 2001; Smith et al., 1998). Hence the development of therapies which disrupt the molecular chaperone machinery may prove to be beneficial. In some conditions (e.g., Alzheimer's disease, prion diseases and Huntington's disease), misfolded proteins can cause protein aggregation resulting in neurodegenerative disorders. Also, misfolded proteins may result in loss of wild type protein function, leading to deregulated molecular and physiological functions in the cell.
HSPs have also been implicated in cancer. For example, there is evidence of differential expression of HSPs which may relate to the stage of tumour progression (Martin et al., 2000; Conroy et al., 1996; Kawanishi et al., 1999; Jameel et al., 1992; Hoang et al., 2000; Lebeau et al., 1991). As a result of the involvement of HSP90 in various critical oncogenic pathways and the discovery that certain natural products with anticancer activity are targeting this molecular chaperone, the fascinating new concept has been developed that inhibiting HSP function may be useful in the treatment of cancer. The first molecular chaperone inhibitor is currently undergoing clinical trials.
HSP90
HSP90 constitutes about 1–2% of total cellular protein, and is usually present in the cell as a dimer in association with one of a number of other proteins (see, e.g., Pratt, 1997). It is essential for cell viability and it exhibits dual chaperone functions (Young et al., 2001). It plays a key role in the cellular stress response by interacting with many proteins after their native conformation has been altered by various environmental stresses, such as heat shock, ensuring adequate protein folding and preventing non-specific aggregation (Smith et al., 1998). In addition, recent results suggest that HSP90 may also play a role in buffering against the effects of mutation, presumably by correcting the inappropriate folding of mutant proteins (Rutherford and Lindquist, 1998). However, HSP90 also has an important regulatory role. Under normal physiological conditions, together with its endoplasmic reticulum homologue GRP94, HSP90 plays a housekeeping role in the cell, maintaining the conformational stability and maturation of several key client proteins. These can be subdivided into three groups: (a) steroid hormone receptors, (b) Ser/Thr or tyrosine kinases (e.g., ERBB2, RAF-1, CDK4, and LCK), and (c) a collection of apparently unrelated proteins, e.g., mutant p53 and the catalytic subunit of telomerase hTERT. All of these proteins play key regulatory roles in many physiological and biochemical processes in the cell. New HSP90 client proteins are continuously being identified.
The highly conserved HSP90 family in humans consists of four genes, namely the cytosolic HSP90α and HSP90β isoforms (Hickey et al., 1989), GRP94 in the endoplasmic reticulum (Argon et al., 1999) and HSP75/TRAP1 in the mitochondrial matrix (Felts et al., 2000). It is thought that all the family members have a similar mode of action, but bind to different client proteins depending on their localization within the cell. For example, ERBB2 is known to be a specific client protein of GRP94 (Argon et al., 1999) and type 1 tumour necrosis factor receptor (TNFR1) and RB have both been shown to be clients of TRAP1 (Song et al., 1995; Chen et al., 1996).
HSP90 participates in a series of complex interactions with a range of client and regulatory proteins (Smith, 2001). Although the precise molecular details remain to be elucidated, biochemical and X-ray crystallographic studies (Prodromou et al., 1997; Stebbins et al., 1997) carried out over the last few years have provided increasingly detailed insights into the chaperone function of HSP90.
The monomer of HSP90 consists of conserved 25 kDa N terminal and 55 kDa C terminal domains joined together by a charged linker region (not present in TRAP1) (Prodromou and Pearl, 2000a). Both the N and C termini of HSP90 are reported to bind to substrate polypeptides including client proteins and co-chaperones. The N terminus contains an unusual ATP binding site that has structural homology with type II topoisomerase gyrase B, an N-terminal fragment of the MutL DNA mismatch repair protein and a C-terminal fragment of the histidine kinase Che A (Prodromou and Pearl, 2000a).
Following earlier controversy on this issue, it is now clear that HSP90 is an ATP-dependent molecular chaperone (Prodromou et al, 1997), with dimerization of the nucleotide binding domains being essential for ATP hydrolysis, which is in turn essential for chaperone function (Prodromou et al, 2000a). Binding of ATP results in the formation of a toroidal dimer structure in which the N terminal domains are brought into closer contact with each other resulting in a conformational switch known as the ‘clamp mechanism’ (Prodromou and Pearl, 2000b).
The function of HSP90 is regulated by association with a number of co-chaperones that combine in various ways to form a series of multimeric protein complexes. Interactions with these various partners in different heterocomplexes may be restricted by temporal, spatial and biochemical factors. A number of these cochaperones contain a tetracopeptide repeat and binding of these proteins to HSP90 has been localised to the C terminal MEEVD motif (Prodromou and Pearl, 2000a).
A number of reviews describe in detail the molecular chaperone role of HSP90 and its importance in the conformational stability and function of the currently identified client proteins (Scheibel et al, 1998; Smith et al., 2001).
Known HSP90 Inhibitors
The first class of HSP90 inhibitors to be discovered was the benzoquinone ansamycin class, which includes the compounds herbimycin A and geldanamycin.

These agents are natural products, initially isolated from actinomycete broths (DeBoer, 1970). However, it was not until the 1980s that their potential application as antitumour agents was discovered. They were shown to reverse the malignant phenotype of fibroblasts transformed by the v-Src oncogene (Uehara et al., 1985), and subsequently to exhibit potent antitumour activity in both in vitro (Schulte et al., 1998) and in vivo animal models (Supko et al., 1995).
Initially, the benzoquinone ansamycins were thought to act as tyrosine kinase inhibitors. However, it has since transpired that depletion of oncogenic protein kinases via the ubiquitin proteasome pathway, rather than inhibition of their catalytic activity, is predominantly responsible for their antitumour activity. Subsequent immunoprecipitation and affinity matrix studies have shown that the major mechanism of action of geldanamycin involves binding to HSP90 (Whitesell et al., 1994; Schulte and Neckers, 1998). Moreover, X-ray crystallographic studies have shown that geldanamycin competes at the ATP binding site and inhibits the intrinsic ATPase activity of HSP90 (Prodromou et al., 1997; Panaretou et al., 1998). This in turn prevents the formation of mature multimeric HSP90 complexes capable of chaperoning client proteins. As a result, the client proteins are targeted for degradation via the ubiquitin proteasome pathway. Recent results suggest that this involves the recruitment of other regulatory proteins—such as the ubiquitin ligase, carboxy terminus of HSC70 interacting protein (CHIP)—to the HSP90 complex (Connell et al., 2001). The particular functions of these HSP90 client proteins and how they may be affected by HSP90 inhibition is discussed in two earlier reviews on HSP90 inhibitors (Neckers et al., 1999; Ochel et al., 2001).
Geldanamycin showed activity in human tumour xenograft models but progression of this compound to clinical trial was halted due to unacceptable levels of hepatotoxicity which was seen at doses required for therapeutic activity (Supko et al, 1995). However, following screening of a range of geldanamycin analogues at the US National Cancer Institute (NCI) it was discovered that 17-allylamino, 17-demethoxygeldanamycin (17AAG) retains the property of HSP90 inhibition resulting in client protein depletion and antitumour activity in cell culture and xenograft models (Schulte et al, 1998; Kelland et al, 1999), but has significantly less hepatotoxicity than geldanamycin (Page et al, 1997). 17AAG is currently being evaluated in Phase I clinical trials using a number of different scheduling regimens under the auspices of the NCI and the UK Cancer Research Campaign (CRC).

A range of geldanamycin analogues has already been described (Schnur et al, 1995a and b), of which 17AAG appeared to be the most promising in terms of therapeutic index. The clinical development of 17AAG and the search for additional analogues that may have improved pharmaceutical properties (e.g., solubility, oral bioavailability) and different pharmacological behaviour (Sybert and Spiegel, 2001) continues. Structure-activity relationships (SAR) with 17AAG analogues have shed more light on the chemical features required for HSP90 inhibitory activity and also for the NQO1 potentiation effect (Schnur et al, 1995a and b, Maloney et al, 1999).
Radicicol is a macrocyclic antibiotic isolated from Monosporium bonorden. It was shown to reverse the malignant phenotype of v-Src and v-Ha-Ras transformed fibroblasts (Kwon et al, 1992; Zhao et al, 1995).

Like the benzoquinone ansamycins, radicicol was at first thought to act as a tyrosine kinase inhibitor. However, it was shown subsequently to degrade a number of signalling proteins as a consequence of HSP90 inhibition (Schulte et al., 1998). X-ray crystallographic data confirmed that radicicol also binds to the N terminal domain of HSP90 and inhibits the intrinsic ATPase activity (Roe et al., 1998). Interestingly, radicicol is more potent at inhibiting HSP90 ATPase activity compared to geldanamycin and 17AAG (Panaretou et al., 1998), even though they have similar growth inhibitory effects on tumour cells. This may be a consequence of differences in the cellular uptake or metabolism of these compounds. Radicicol binds to all HSP90 family members, although it has a weaker binding affinity to both GRP94 and TRAP1 than the cytosolic HSP90 isoforms (Schulte et al., 1999).
However, like 17AAG, the structure of radicicol has a number of adverse pharmacological properties that could lead to unfavourable metabolism. These include an epoxide residue, keto group, two phenolic hydroxyl groups and Michael acceptor. Radicicol lacks antitumour activity in vivo due to the unstable chemical nature of the compound. Oxime derivatives of radicicol (KF25706 and KF58333) have been synthesised which retain the HSP90 inhibitory activity of radicicol, and KF25706 has been shown to exhibit in vivo antitumour activity in human tumour xenograft models (Soga et al., 1999).
Coumarin antibiotics are known to bind to bacterial DNA gyrase at an ATP binding site homologous to that of the HSP90. The coumarin, novobiocin, was shown to bind to the carboxy terminus of HSP90, i.e., at a different site to that occupied by the benzoquinone ansamycins and radicicol which bind at the N-terminus (Marcu et al., 2000b). However, this still resulted in inhibition of HSP90 function and degradation of a number of HSP90-chaperoned signalling proteins (Marcu et al., 2000a). Geldanamcyin cannot bind HSP90 subsequent to novobiocin; this suggests that some interaction between the N and C terminal domains must exist and is consistent with the view that both sites are important for HSP90 chaperone properties.

A purine-based HSP90 inhibitor, PU3, has been synthesized based on rationale drug design with the aid of the X-ray crystal structure (Chiosis et al., 2001). This agent was shown to result in the degradation of signalling molecules, including ERBB2, and to cause cell cycle arrest and differentiation in breast cancer cells (Chiosis et al., 2001). Although less potent than 17AAG, it is more soluble and so may be formulated in more conventional vehicles and could potentially have more favourable oral bioavailability.

HSP90 as a Therapeutic Target
Due to its involvement in regulating a number of signalling pathways that are crucially important in driving the phenotype of a tumour, and the discovery that certain bioactive natural products exert their effects via HSP90 activity, the molecular chaperone HSP90 is currently being assessed as a new target for anticancer drug development (Neckers et al., 1999).
The predominant mechanism of action of geldanamycin, 17AAG, and radicicol involves binding to HSP90 at the ATP binding site located in the N-terminal domain of the protein, leading to inhibition of the intrinsic ATPase activity of HSP90 (see, e.g., Prodromou et al., 1997; Stebbins et al., 1997; Panaretou et al., 1998).
Inhibition of HSP90 ATPase activity prevents recruitment of co-chaperones and encourages the formation of a type of HSP90 heterocomplex from which these client proteins are targeted for degradation via the ubiquitin proteasome pathway (see, e.g., Neckers et al., 1999; Kelland et al., 1999).
Treatment with HSP90 inhibitors leads to selective degradation of important proteins involved in cell proliferation, cell cycle regulation and apoptosis, processes which are fundamentally important in cancer.
Inhibition of HSP90 function has been shown to cause selective degradation of important signalling proteins involved in cell proliferation, cell cycle regulation and apoptosis, processes which are fundamentally important and which are commonly deregulated in cancer (see, e.g., Hostein et al., 2001). An attractive rationale for developing drugs against this target for use in the clinic is that by simultaneously depleting proteins associated with the transformed phenotype, one may obtain a strong antitumour effect and achieve a therapeutic advantage against cancer versus normal cells. These events downstream of HSP90 inhibition are believed to be responsible for the antitumour activity of HSP90 inhibitors in cell culture and animal models (see, e.g., Schulte et al., 1998; Kelland et al., 1999).
Khilya et al., 1994, describe the synthesis of a number of 3,4-diaryl pyrazoles (see the following table), by reaction of isoflavones with benzodioxolane (n=1), benzodioxane (n=2), or benzodioxepane (n=3) upon boiling in alcohol with hydrazine hydrate. However, nowhere in the document is there provided any teaching of possible uses of these compounds.
TABLE 1Compounds in Khilya et al., 1994 CompoundRR1R2nR32aHEtH1H2bHPrH1H2cMeEtH1H2dHHH2H2eHEtH2H2gHPrH2(*)H2hMeHH2H2iMeEtH2H2jMePrH2H2kHEtMe1H2lHPrMe1H2mHHMe2H2nHEtMe2H2oHPrMe2H2pHPrMe3H3HPrMe3Me(*) based on the empirical formula in Table 1 therein, and the fact that this compound would otherwise be identical to 2b.
Penning et al., 1997, describe various 3,4-diarylpyrazoles (see the following table) which apparently are potent and selective inhibitors of cyclooxygenase-2 (COX-2), some of which apparently have anti-inflammatory activity.
TABLE 2Compounds in Penning et al., 1997 CompoundR1R2R3 4—H—H—Me 5—CF3—H—Me6a—H—CH2CH2═CH2—Me6b—H—CH2CH2Ph—Me7a—CF3—CH2CH2═CH2—Me7b—CF3—CH2CH2Ph—Me7c—CF3—Et—Me7d—CF3—CH2CO2Et—Me7e—CF3—CH2CONHPh—Me10—CF3—Et—NH2
Although HSP90 inhibitors are known, there remains a great need for potent HSP90 inhibitors which offer one or more of the following advantages:                (a) improved activity.        (b) improved selectivity (e.g., against tumour cells versus normal cells).        (c) complement the activity of other treatments (e.g., chemotherapeutic agents);        (d) reduced intensity of undesired side-effects;        (e) fewer undesired side-effects;        (f) simpler methods of administration;        (g) reduction in required dosage amounts;        (h) reduction in required frequency of administration;        (i) increased ease of synthesis, purification, handling, storage, etc.;        (j) reduced cost of synthesis, purification, handling, storage, etc.        
Thus, one aim of the present invention is the provision of compounds which are potent HSP90 inhibitors, anticancer agents, etc. which offer one or more of the above properties and advantages.
The inventors have discovered that certain 3,4-diarylpyrazoles, described herein, offer one or more of the above properties and advantages, and additionally are surprisingly and unexpectedly more active than many of the corresponding known analogues.
The present invention pertains to certain 3,4-diarylpyrazoles, described herein, and the discovery of their surprising and unexpected activity as HSP90 inhibitors.